The central hypothesis of this application is AMPK maintains the balance of mitochondrial fission and fusion by promoting the autophagic degradation of DRP1. This hypothesis will be tested in the following specific aims. Aim 1 is to establish the essential role of AMPK in maintaining the balance between mitochondrial fission and fusion. In this aim, we will generate the mice with endothelium-specific overexpression of constitutively active AMPK?1 (TG-CA- AMPK). We will use these mice to determine whether constitutive AMPK activation in ECs alleviates mitochondrial fission and endothelial dysfunction in vivo. To determine whether inhibition of endothelial AMPK activity increases endothelial dysfunction and mitochondrial fission in the endothelium, we will examine whether genetic deletion of AMPK promotes mitochondrial fission and endothelial dysfunction in vivo using mice with endothelium-specific deletion of AMPK (AMPK?1endo-/- or AMPK?2endo-/-). We will test whether constitutive AMPK activation in ECs prevents diabetes-induced mitochondrial fission and endothelial dysfunction in vivo by using streptozotocin (STZ) to induce diabetes in wild-type (WT) and TG-CA-AMPK mice. In these mice, we will observe AMPK activity and mitochondrial fission in the development of endothelial dysfunction. Aim 2 is to elucidate the molecular mechanisms by which AMPK inhibits DRP1-dependent mitochondrial fission in ECs. In this aim, we establish the role of AMPK in regulating autophagy and DRP1 expression in ECs. To this end, we will (a) examine if AMPK activation enhances autophagic activity and prevents DRP1 accumulation in diabetic TG-CA-AMPK mice and (b) test whether inhibition of AMPK suppresses autophagy and aggravates diabetes-enhanced DRP1 protein levels in AMPK?1endo-/- or AMPK?2endo-/- mice. (ii) To define the mechanism underlying the autophagy-mediated degradation of DRP1, we will manipulate autophagic activity and measure DRP1 expression. This will allow us to examine if p62 can mediate the autophagic degradation of DRP1, determine the interaction between DRP1 and p62, and identify the region of p62 that is required for the interaction with DRP1. (iii) To determine whether AMPK activation promotes DRP1 degradation via the autophagic pathway, we will treat diabetic WT and heterozygous beclin1 mice (beclin1+/-, with reduced autophagy) with the AMPK activator, A769662, and examine whether inhibition of autophagy can attenuate the AMPK-promoted degradation of DRP1.